The industrial cultivation of fish through aquaculture is expanding dramatically. Infectious diseases caused by pathogens constitute a major problem that the industrial aquatic establishments must overcome. To prevent the onset of these infectious diseases, the recurrent strategy is the application of vaccines. Nearly all of the vaccines currently in use in aquaculture correspond to live-attenuated or inactivated cultures of the same pathogenic organism that produces the disease. Recombinant vaccines are increasingly growing at the research laboratory level, but field application is still almost exclusively through injection techniques.
There are two ways in which fish can be vaccinated: the vaccines can be applied by injection or by immersion. At present, one of the biggest problems in aquaculture is that the injectable vaccines are difficult and expensive to administer, and generally cannot be used in young weighing less than 15-20 grams due both to the risk of producing intra-peritoneal adhesions and to the production of high dispersion in fish sizes at harvesting. Additionally, use of injectable vaccines causes stress in the fish, high cost of labor, and risk of accidents from inadvertent injections to the users.
In spite of the potential advantages of immersion vaccines, it has been the injectable vaccines that have had a greater development in commercial aquaculture. The intraperitoneal injection of fish is currently the most common method to administer vaccines, principally due to its high efficiency and meticulous dosing that guarantees adequate plasma levels. Current immersion vaccines are often characterized by their inefficient absorption through the fish's skin and can suffer from reduced immunogenicity. As a result, highly immunogenic compositions such as live-attenuated or inactivated pathogens are generally required.
Flavobacterium psychrophilum is a gram-negative bacterial fish pathogen that causes bacterial coldwater disease (CWD), and is considered to be an important pathogen affecting salmonid aquaculture due to its wide distribution and economic impact. In the United States, it is estimated that annual losses incurred from CWD in the Pacific Northwest alone are approximately 9.6 and 4 million dollars for commercial aquaculture of rainbow trout (Oncorhynchus mykiss Walbaum) and conservation aquaculture of salmonid species, respectively.
Flavobacterium columnare is an aquatic bacterium that is highly infectious for both warm and cold water species of fish. In the channel catfish (Ictalurus punctatus), it is the causative agent of columnaris disease. Flavobacterium columnare is a Gram-negative, rod shaped, pathogen that has been isolated from channel catfish in areas of the southeastern United States where this species is cultured. The disease also affects sports fish (i.e., walleye and largemouth bass) and aquarium fishes. Medicated feed (antibiotics) is currently used to try to control this bacterial infection. However, these treatments are limited in their effectiveness, and most producers have discontinued use of medicated feeds. Prevention of columnaris disease by vaccination is an important goal, and a top priority of catfish and other fish producers throughout the world. Estimated savings to these industries would be in excess of $100 million annually.
Treatment options for Flavobacterium are limited, and include reducing pathogen concentrations, eliminating the spread of the pathogen, and the use of antibiotics. However, the effectiveness of treatment is usually inconsistent, and there are potential risks of developing antibiotic-resistant strains. Therefore, a vaccine to prevent infection is desired.
Infectious pancreatic necrosis virus (IPNV) is the causal agent of a highly contagious and destructive disease of Rainbow and Brook trout and Atlantic salmon. Highly virulent strains of IPNV may cause greater than 90% mortality in hatchery stocks less than four months old. Survivors of infection can remain lifelong asymptomatic carriers and serve as reservoirs of infection, shedding virus in their feces and reproductive products. Therefore, IPNV is a pathogen of major economic importance to the aquaculture industry.
IPNV is the prototype of the Birnaviridae virus family. IPNV contains a bisegmented dsRNA genome, which is surrounded by a single-shelled icosahedral capsid. The larger of the two genome segments, segment A (3097 bases), encodes a 106-kDa precursor polyprotein which is processed to yield mature viral structural proteins VP2 and VP3, and VP4 (also named NS) a non-structural protein (Duncan et al. 1987). VP2 has been identified as the major host protective antigen of IPNV.
An ideal vaccine for IPNV must induce protection at an early age, prevent carrier formation, and should be effective against a large number of IPNV subtypes. Inactivated IPNV vaccines have been found to be efficacious by intraperitoneal inoculation IPNV (described in US2003072772 and U.S. Pat. No. 8,168,201, hereby incorporated by reference); however, they are not efficient and result in additional dosing through injection. U.S. Pat. No. 8,168,201 describes antigenic compositions comprising particular antigens as subunit vaccines wherein the vaccines can further comprise additional promiscuous T-cell epitopes.
Tenacibaculum maritimum (formerly, Cytophaga marina, Flexibacter marinus and F. maritimus) is the causative agent of flexibacteriosis in marine fish and belongs to the Flavobacteriaceae family of bacteria (Wakabayashi et al., 1986; Bernardet and Grimont, 1989; Sukui et al., 2001). Marine flexibacteriosis is widely distributed in cultured and wild fish in Europe, Japan, North America and Australia (McVicar and White, 1979, 1982). Among the cultured fish, the disease has been reported in turbot, sole, gilthead seabream, seabass, red seabream, black seabream (Acanthopagrus schlegeli), flounder and salmonids.
Although both adults and juveniles may be affected by marine flexibacteriosis, younger fish suffer a more severe form of the disease. An increased prevalence and severity of the disease has been reported at higher temperatures. In addition to water temperature, the disease is influenced by a multiplicity of environmental (stress) and host-related factors (skin surface condition). In general, the affected fish have an eroded and haemorrhagic mouth, ulcerative skin lesions, frayed fins and tail rot. A systemic disease can be also established involving different internal organs. The loss of epithelial fish surface, typical of this disease, is also a portal of entry for other bacterial or parasitic pathogens.
In order to be commercially useful, a vaccine for fish must be capable of conferring protective immunity against a pathogen when the vaccine is administered by practical methods, such as immersing the fish in water containing the vaccine. Vaccination protocols that require individual handling of fish, such as by injection, are not practical for many commercial aquaculture operations.
U.S. Patent Publication 2008/0317781 to Cain et al. provides that “immunization with killed bacteria has been attempted with F. psychrophilum, and protection obtained by immersion or by injection with the killed bacteria has been minimal.” Consequently, Cain et al. produced a novel live attenuation process for F. psychrophilum that purportedly produced effective immunization through immersion techniques.
To date, no effective commercial immersion vaccines exist based on subunit antigens, however, particularly with recombinant antigens. In view of the concerns raised by Cain et al. relating to the difficulty producing immunogenic immersion vaccines with killed bacteria, subunit immersion vaccines would presumably face even greater challenges. Against this backdrop, Applicants approached the difficult task of developing a subunit immersion vaccine.